HOMOLOGY MODELLING

Ana Rojas Mendoza.

ProteinDesign Group.
Centro Nacional de Biotecnologia (C.N.B.- C.S.I.C.)
Campus Universidad Autonoma.Cantoblanco. 28049 Madrid.
Tlf: +34-91-5854570. Fax: +34-91-5854506.
e-mail: arojas@gredos.cnb.uam.es

Madrid-Feb-04

THE PRESENTATION of the course.

You'll find here

1.- Servers for homology Modeling

2.- Homology modeling EXAMPLES

3.- Practical examples for Homology modeling

4.- Quick Guide for homology modeling

SERVERS

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SWISS-MODELSWISS-MODEL is an automated comparative modelling server (ExPASy, CH)
PHmodelsServer using homology modelling (BioCentrum, Denmark)
SDSC1Protein structure homology modeling server (San Diego, USA)
3D-JIGSAWAutomated system for 3D models for proteins (Cancer Research UK)
WHATIFWHAT IF Web interface: homology modelling, drug docking, electrostatics calculations, structure validation and visualisation.

A HOMOLOGY MODELING EXAMPLE

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1.-THIS IS THE TARGET SEQUENCE:

>target sequence

TQALVTLTSACADSSPSCDLFKNQGYCGEKFFDWMRKNCAKTCGFCGTGGGAETGGSDGVCGKTSVQQSR
IISGTNARPGAWPWMASLYMLSRSHICGGSLLNSRWILTASHCVVGTGATTKNLVIKLGEHDHYDKDGFE
QQFDVEKIIPHPAYKRGPLKNDIALIKLKTPARINKRVKTICLPKKGSAPSVGSRECYLAGWGSIRHPGG
SYHTLQQAMLPVVSYTNCHNQKNFVCAGFGKSSLTNACRGDSGGPLMCRKSDGSWEQHGIASFVVEYCKY
YTAFTPVANYIDWINQHINK

2.- Now: Submit the sequence to the SWISS-MODEL prgram:

We need to use the "First approach mode", input your name and e-mail address SWISS-MODEL(First Approach mode).
After submission you receive a confirmation message (and a confirmatory e-mail= SEE an example of it! ):
======================================================================
SWISS-MODEL Submission

Your request submission was successful.
You will be notified when the server starts working on it.
All further communications from SWISS-MODEL will come via E-mail.

SWISS MODEL http://swissmodel.expasy.org/

Torsten Schwede , Jürgen Kopp, Nicolas Guex, & Manuel C. Peitsch

REQUEST SUMMARY
Return address:	arojas@cnb.uam.es
Sequence:	TQALVTLTSACADSSPSCDLFKNQGYCGEKFFDWMRKNCAKTCGFCGTGGGAETGGSDGVCGKTSVQQSR IISGTNARPGAWPWMASLYMLSRSHICGGSLLNSRWILTASHCVVGTGATTKNLVIKLGEHDHYDKDGFE QQFDVEKIIPHPAYKRGPLKNDIALIKLKTPARINKRVKTICLPKKGSAPSVGSRECYLAGWGSIRHPGG SYHTLQQAMLPVVSYTNCHNQKNFVCAGFGKSSLTNACRGDSGGPLMCRKSDGSWEQHGIASFVVEYCKY YTAFTPVANYIDWINQHINK
Title:	sp_target
Output Format	Spdbv
WhatCheck Report	No
Foldrecognition	Never
PredictProtein	Never
Mail Attachment	Yes

====================================

You'll receive an e-mail with all the proccesses conducted by SWISS-MODEL

SWISS-MODEL search for structural homologs. It uses "BLASTP2" in "ExNRL-3D" Database. Results are shown according to P(N) from BLAST

Found 1bruP.pdb with P(N)=2e-40

Found 4chaB.pdb with P(N)=5e-38

Found 1choE.pdb with P(N)=5e-38

Found 1gmh_.pdb with P(N)=6e-38

Found 8gch_.pdb with P(N)=6e-38

Found 1gcd_.pdb with P(N)=6e-38

Found 1pytC.pdb with P(N)=6e-38

Found 3gch_.pdb with P(N)=8e-38

Found 5gch_.pdb with P(N)=8e-38

Found 4gch_.pdb with P(N)=8e-38

Found 1gmcA.pdb with P(N)=8e-38

Found 2gmt_.pdb with P(N)=8e-38

........

(cutfor clarity)

With the "SIM" program all the "templates" (3D structures usedto model) with an id% > 25% more than 25 residues lenght will be retrieved. In addition, this program can recognize different domains. This process is reflected here...:

1bruP.pdb:44.98 % identity

4chaB.pdb:36.14 % identity

1choE.pdb:36.14 % identity

1gmh_.pdb:36.6 % identity

8gch_.pdb:36.6 % identity

1gcd_.pdb:36.6 % identity

1pytC.pdb:40.96 % identity

3gch_.pdb:30.78 % identity

5gch_.pdb:30.78 % identity

1bruP.pdb| --------------------------------------

4chaB.pdb| ---------------------------------------

1choE.pdb| ---------------------------------------

1gmh_.pdb| ---------------------------------------

8gch_.pdb| ---------------------------------------

1gcd_.pdb| ---------------------------------------

1pytC.pdb| --------------------------------------

3gch_.pdb| ---------------------------------------

5gch_.pdb| ---------------------------------------

4gch_.pdb| ---------------------------------------

1gmcA.pdb| ---------------------------------------

2gmt_.pdb| ---------------------------------------

etc etc...............................

1a0lA.pdb 1a0lB.pdb 1a0lC.pdb 1a0lD.pdb 1a3bH.pdb 1a3eH.pdb 1a61H.pdb 1abiH.pdb

1acbE.pdb 1ad8H.pdb 1ae8H.pdb 1afeH.pdb 1ahtH.pdb 1ai8H.pdb 1aixH.pdb 1amhA.pdb
-------- ETC etc..

Now batch files are created for ProMod to generate a model

Creating Batch files for ProMod (if any):
	Batch.1: residues 56 - 300 of submitted sequence.

Exiting AlignMaster


ProModII trace log for Batch.1
============================================================

ProModII: 3.70 (SP3)
ProModII: Loading Template: 1bruP.pdb
ProModII: Loading Template: 4chaB.pdb
ProModII: Loading Template: 1choE.pdb
ProModII: Loading Template: 1gmh_.pdb
ProModII: Loading Template: 8gch_.pdb
ProModII: Loading Raw Sequence
ProModII: Iterative Template Fitting
ProModII: Iterative Template Fitting
ProModII: Iterative Template Fitting
ProModII: Iterative Template Fitting
ProModII: Generating Structural Alignment
ProModII: Aligning Raw Sequence
ProModII: Refining Raw Sequence Alignment
ProModII: ProModII: doing complex assignment of backbone
ProModII: N-terminal overhang trimmed for chain ' '. Start at residue: 16
ProModII: ProModII: adding blocking groups
ProModII: Weighting Backbones
ProModII: Src residue is not amino-acid
ProModII: Src residue is not amino-acid
ProModII: Averaging Sidechains
ProModII: Adding Missing Sidechains
ProModII: Trying Ligating with anchor residues SER 32 and MET 35
ProModII: Trying Ligating with anchor residues SER 32 and LEU 36
ProModII: connectivity problem --> including residue LEU 22
ProModII: Trying Ligating with anchor residues SER 32 and SER 37
ProModII: connectivity problem --> including residue SER 23
ProModII: Trying Ligating with anchor residues SER 32 and ARG 38
ProModII: connectivity problem --> including residue ARG 24
ProModII: Trying Ligating with anchor residues SER 32 and SER 39
ProModII:  Number of Ligations found: 500
ProModII: ACCEPTING loop  378: clash=   0 FF=        195.4 PP= -7.00
ProModII: Small Ligation (C-N < 3.0A) ignored;
ProModII: GROMOS will repair it at residue SER 25
ProModII: connectivity problem (C-N > 3.0A) at residue: 71
ProModII: Trying Ligating with anchor residues GLY 83 and GLN 86
ProModII:  Number of Ligations found: 3
ProModII: all loops are bad; continuing CSP with larger segment
ProModII: Trying Ligating with anchor residues ASP 82 and GLN 86
ProModII:  Number of Ligations found: 22
ProModII: all loops are bad; continuing CSP with larger segment
ProModII: Trying Ligating with anchor residues LYS 81 and GLN 86
ProModII:  Number of Ligations found: 500
ProModII: ACCEPTING loop  230: clash=   0 FF=        -17.5 PP= -2.00
ProModII: connectivity problem (C-N > 3.0A) at residue: 91
ProModII: Trying Ligating with anchor residues PRO 103 and ASN 106
ProModII:  Number of Ligations found: 12
ProModII: all loops are bad; continuing CSP with larger segment
ProModII: Trying Ligating with anchor residues GLY 102 and ASN 106
ProModII:  Number of Ligations found: 14
ProModII: all loops are bad; continuing CSP with larger segment
ProModII: Trying Ligating with anchor residues GLY 102 and ASP 107
ProModII: connectivity problem --> including residue ASP 93
ProModII: Trying Ligating with anchor residues GLY 102 and ILE 108
ProModII: Trying Ligating with anchor residues ARG 101 and ILE 108
ProModII:  Number of Ligations found: 273
ProModII: ACCEPTING loop  218: clash=   0 FF=       1486.4 PP=  0.00
ProModII: Trying Ligating with anchor residues ASN 175 and ASN 178
ProModII: Trying Ligating with anchor residues HIS 174 and ASN 178
ProModII:  Number of Ligations found: 22
ProModII: ACCEPTING loop   10: clash=   0 FF=       -294.1 PP= -3.00
ProModII: Trying Ligating with anchor residues GLY 183 and LYS 186
ProModII: Trying Ligating with anchor residues ALA 182 and LYS 186
ProModII: Trying Ligating with anchor residues ALA 182 and SER 187
ProModII: Trying Ligating with anchor residues CYS 181 and SER 187
ProModII: Trying Ligating with anchor residues CYS 181 and SER 188
ProModII: Trying Ligating with anchor residues VAL 180 and SER 188
ProModII:  Number of Ligations found: 500
ProModII: all loops are bad; continuing CSP with larger segment
ProModII: Trying Ligating with anchor residues VAL 180 and LEU 189
ProModII:  Number of Ligations found: 112
ProModII: all loops are bad; continuing CSP with larger segment
ProModII: Trying Ligating with anchor residues PHE 179 and LEU 189
ProModII:  Number of Ligations found: 500
ProModII: all loops are bad; continuing CSP with larger segment
ProModII:  +++ Warning: Ligation Failed, SparePart will be inserted later 
ProModII:  +++          It is usually the sign that the region is misaligned.
ProModII: connectivity problem (C-N > 3.0A) at residue: 177
ProModII: Trying Ligating with anchor residues LEU 189 and ALA 192
ProModII: Trying Ligating with anchor residues SER 188 and ALA 192
ProModII: Trying Ligating with anchor residues SER 187 and ALA 192
ProModII: Trying Ligating with anchor residues LYS 186 and ALA 192
ProModII: Trying Ligating with anchor residues GLY 185 and ALA 192
ProModII: Trying Ligating with anchor residues PHE 184 and ALA 192
ProModII: Trying Ligating with anchor residues GLY 183 and ALA 192
ProModII: Trying Ligating with anchor residues ALA 182 and ALA 192
ProModII:  +++ Warning: Ligation Failed, SparePart will be inserted later 
ProModII:  +++          It is usually the sign that the region is misaligned.
ProModII: connectivity problem (C-N > 3.0A) at residue: 207
ProModII: Trying Ligating with anchor residues VAL 219 and TYR 222
ProModII:  Number of Ligations found: 7
ProModII: ACCEPTING loop    0: clash=   0 FF=        -37.3 PP=  1.00
ProModII: Trying Ligating with anchor residues LYS 224 and THR 227
ProModII:  Number of Ligations found: 2
ProModII: ACCEPTING loop    1: clash=   0 FF=        261.7 PP=  2.00
ProModII: Building CSP loop with anchor residues VAL 59 and ALA 64
ProModII:  Number of Ligations found: 53
ProModII: all loops are bad; continuing CSP with larger segment
ProModII: Building CSP loop with anchor residues VAL 59 and THR 65
ProModII:  Number of Ligations found: 119
ProModII: ACCEPTING loop  114: clash=   0 FF=        560.6 PP=  1.00
ProModII: Building CSP loop with anchor residues SER 136 and SER 139
ProModII: Building CSP loop with anchor residues PRO 135 and SER 139
ProModII: Building CSP loop with anchor residues ALA 134 and SER 139
ProModII:  Number of Ligations found: 500
ProModII: ACCEPTING loop   22: clash=   0 FF=        -38.9 PP= -2.00
ProModII: Finding Spare-Part loop with anchor residues ASN 178 and ASN 191
ProModII: connectivity problem --> including residue ASN 177
ProModII: Finding Spare-Part loop with anchor residues ASN 178 and ALA 192
ProModII: ACCEPTING loop   1 from 4RHV1  Clash=   4 FF=        555.6 PP=-29.69
ProModII: BadPhi=   1 BadGX=   0 BadXP=   0 weakXP=   0 Score= 7.00  rms= 0.00
ProModII: Optimizing Sidechains
ProModII: Dumping Preliminary Model
ProModII: Adding Hydrogens
ProModII: Optimizing loops and OXT (nb = 41)
ProModII: Final Total Energy:        5009.919 KJ/mol
ProModII: Removing Hydrogens
ProModII: Fixing Atom Nomenclature
ProModII: Dumping Sequence Alignment
***

Getting the 3D coordinates from the model

Finally you'll receive an e-mail with the pdb coordinates if your model.

See in the image the model (RED) and one target (1bruP) in grey.
It must be mentioned that coordinates are generated via predictions, so any conclusion must be taken carefully. (HERE Access to an e-mail with the 3D coordinates.). Note that several templates have been selected to generate the model although in the figure only one is represented.

An Example: Swiss Pdb-Viewer to visualize the model

FTSA_ECOLI_seq.txt

QUERY SEQUENCE

Blast2_PDB

[DEMO]

PDB OCA Browser

[DEMO]

1e4f.pdb	3D coordinates of FtsA (Apo Form) from Thermotoga Maritima
FTSA_ECOLI_Tracelog.html	SwissModel TraceLog AAAa010Mt
FTSA_ECOLI_WhatCheck.html	SwissModel WhatCheck AAAa010Mt Batch.0
AAAa010Mt.pdb	THEORETICAL MODEL
1e4f_WhatCheck.html	WHAT IF Check report: Verification log for 1E4F. (ver PDBsum)

TRAINING SECTIONS :

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PRACTISE 1:

Why is the following enzyme inactive (hypothetical mutant)?

Sequence:

ANFQYIITEKKGKNSSVGLIQLNRPKALNALCNGLIEELNQALETFEEDP

AVGAIVLTGGEKAFAAGWDIKEMQNRTFQDCYSGKFLSHWDHITRIKKPV

IAAVNGYALGGGCELAMMCDIIYAGEKAQFGQPEILLGTIPGAGGTQRLT

RAVGKSLAMEMVLTGDRISAQDAKQAGLVSKIFPVETLVEEAIQCAEKIA

NNSKIIVAMAKESVNAAFEMTLTEGNKLEKKLFYSTFATDDRREGMSAFV

EKRKANFKDH

Steps to follow:

Use BLAST to identify the sequence
Check out SWISSPROT (Pfam, InterPro, etc...) to get info about the biochemistry of the enzyme.
Check out possible structure of this protein (the wild type).
Use the server SwissModel to generate a model of the mutant.
Visualize both structures to find distintive features which might explain the loss of enzymatic activity.

Output files (ONLY USE IF YOU CAN'T CONNECT TO SWISS-MODEL!!!)

Welcome file: Swiss_Welcome.txt
Tracelog: Swiss_Tracelog.txt
Template PDB: 1dub.pdb
Template chain E PDB: 1DUBE_WT.pdb
Generated model for mutant PDB: 1DUBE_A98W.pdb

OTHER EXCERSISES (use SwissModel server):

HYPOTHETICAL 13.1 KD PROTEIN IN ILV6-CWH36 INTERGENIC REGION.

>Sequence:

MWVPSMYPVKPPFISINLENFDMNTISSSLPIQEYIDSNGWIALPILHCWDPAAMNLIMV

VQELMSLLHEPPQDQAPSLPPKPNTQLQQEGEYSPTAPKAQVSTPKTAIASTSTTSTGI

"pacC"; product: "DNA binding protein".

Sequence:

MLGAMAEEAVAPVAVPTTQEQPTSQPAAAQVTTVTSPSVTATAAAATAAVASPQANGNAA

SPVAPASSTSRPAEELTCMWQGCSEKLPTPESLYEHVCERHVGRKSTNNLNLTCQWGSCR

TTTVKRDHITSHIRVHVPLKPHKCDFCGKAFKRPQDLKKHVKTHADDSVLVRSPEPGSRN

PDMMFGGNGKGYAAAHYFEPALNPVPSQGYAHGPPQYYQAHHAPQPSNPSYGNVYYALNT

GPEPHQASYESKKRGYDALNEFFGDLKRRQFDPNSYAAVGQRLLSLQNLSLPVLTAAPLP

EYQAMPAPVAVASGPYGGGPHPAPAYHLPPMSNVRTKNDLINIDQFLQQMQDTIYENDDN

VAAAGVAQPGAHYIHNGISYRTTHSPPTQLPSAHATTQTTAGPIISNTSAHSPSSSTPAL

TPPSSAQSYTSGRSPISLPSAHRVSPPHESGSSMYPRLPSATDGMTSGYTAASSAAPPST

LGGIFDNDERRRYTGGTLQRARPASRAASESMDLSSDDKESGERTPKQISASLIDPALHS

GSPGEDDVTRTAKAATEVAERSDVQSEWVEKVRLIEYLRNYIANRLERGEFSDDSEQEQD

QEQEQDQEQEQDQEQGQDRVSRSPVSKADVDMEGVERDSLPRSPRTVPIKTDGESAEDSV

MYPTLRGLDEDGDSKMPS

QUICK GUIDES

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A QUICK GUIDE TO ANALYSE SWISS MODEL RESULTS

EXAMPLE OF QUALITY CHECKS:WHATIF &PROSA

PROTEIN MODELLING AND EVALUATION:

Comparativeprotein modelling : framework, loops, side chains, rotamers

[within Principles of Protein Structure , Comparative Protein Modelling and Visualisation (Nicolas Guex and Manuel C. Peitsch)]

Professional gambling (G. Vriend). A review of homology modeling all the way from template detection till energy refinement.

The use of position specific rotamers in model building by homology